Imaging of microbial interactions has so far been based on well-established electron microscopy methods. This study presents a new way to study bacterial colonies and interactions between bacteria and their viruses, bacteriophages (phages), in situ on agar plates using helium ion microscopy (HIM). In biological imaging, HIM has advantages over traditional scanning electron microscopy with its sub-nanometer resolution, increased surface sensitivity, and the possibility to image nonconductive samples. Furthermore, by controlling the He beam dose or by using heavier Ne ions, the HIM instrument provides the possibility to mill out material in the samples, allowing for subsurface imaging and in situ sectioning. Here, the first HIM-images of bacterial colonies and phage-bacterium interactions are presented at different stages of the infection as they occur on an agar culture. The feasibility of neon and helium milling is also demonstrated to reveal the subsurface structures of bacterial colonies on agar substrate, and in some cases also structure inside individual bacteria after cross-sectioning. The study concludes that HIM offers great opportunities to advance the studies of microbial imaging, in particular in the area of interaction of viruses with cells.
Keywords: bioimaging; focused ion beam milling; helium ion microscopy; phage-bacteria interactions.
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